Considerable interest has been focused on the development of ultrasensitive DNA biosensors following the completion of the Human Genome Project. These biosensors have a wide variety of potential applications that range from genotyping to molecular diagnostics. The use of fluorescently labeled oligonucleotides in conjunction with surface modification techniques affords high density DNA arrays for analyzing specific DNA sequences and gene expression, but only a few of the fluorescence-based techniques have sufficient sensitivities for the detection of DNA at sub-nanomolar levels.
Other transduction techniques, such as autoradiographic, electrochemical, chemiluminescent, and transitional metal-bipyridine complex-based electrochemiluminescentmethods (ECL) have therefore been proposed for ultrasensitive detection of DNA hybridization events. Among them, ECL has been demonstrated to be one of the most sensitive techniques. ECL is the process of generating excited states in a photoactive molecule at an electrode surface, leading to luminescence upon return to the ground state. One compound that has been extensively studied is tris(2,2′-bipyridine)ruthenium [Ru(bpy)32+]. ECL of Ru(bpy)32+ was reported by Tokel et al. some thirty years ago (Tokel et al., J. Am Chem. Soc. 1972, 94, 2862-2866). Because of its low-lying metal-to-ligand charge-transfer (MLCT) excited states, high emission quantum yields (˜4.2% in H2O) and long excited-state lifetimes (˜600 ns), the Ru(bpy)32+/tri-n-propylamine (TPA) system is usually adopted in analytical applications. ECL as a DNA detection technique has the potential to match or exceed the sensitivity of autoradiography since it enjoys the benefit of having different forms of energy for excitation and detection. The key to the ultrahigh sensitivity of ECL lies in its ultralow background noise, which is a direct consequence of having two different forms of energy for analytical signal generation and detection. Unlike fluorescence-based techniques, ECL does not involve an excitation light source and it can theoretically produce a “zero” background.
A promising approach toward the enhancement of the ECL signal is to build up multiple ECL tags on a single double stranded nucleic acid molecule (e.g. DNA). This strategy has the advantage of providing multiple redox sites, thereby greatly increasing the number of charge recombination events per target DNA molecule, and consequently enhancing the sensitivity and detection limit of a DNA biosensor. Two fundamental issues that need to be addressed in the development of multiple ECL tag systems are (i) accessibility of the ECL redox sites to the electrode and to active TPA species, and (ii) electronic independence of the redox sites to avoid intramolecular energy transfer from the excited site to a lowest-lying unoccupied molecular orbital of an acceptance site. It has been demonstrated that as little as 1.0 fM DNA is detected when Ru(bpy)32+ doped polystyrene microspheres are used as ECL labels. The microspheres were shown to be beneficial both for target DNA immobilization and for amplifying ECL signal.
Threading intercalators are an important group of compounds that interact reversibly with modified or unmodified double stranded nucleic acid polymers, such as ds-DNA, ds-peptide nucleic acids, and peptide nucleic acid-nucleic acid hybrids. Some of the known threading intercalators are valuable antitumor drugs currently used for the treatment of ovarian and breast cancers. Threading intercalators share common structural features such as the presence of planar polyaromatic systems, which bind to ds-DNA by insertion between base pairs. It has also been shown that the use of an electroactive DNA intercalator as a hybridization indicator avoids labelling of the target DNA, as is commonly done in conventional DNA detection techniques, which avoids tedious labelling procedures and the use of expensive equipment. These biosensors, however, have to solve a low signal/noise ratio problem since most threading intercalators bind not only to ds-DNA but also, although to a much less extent, to single-stranded DNA (ss-DNA) molecules by electrostatic interaction. Intercalators offering better discrimination between ss- and ds-DNA are being developed for achieving greater signal/noise ratio.